Coxiella Burnetii and Rickettsiella Grylli Genome Project

Goals

The goal of this project is to generate complete genome sequence data from four Coxiella burnetii isolates and Rickettsiella grylli in order to define variations in strains that are responsible for differences in virulence and host range. Additionally, comparative genomics of a newly-discovered Coxiella sp. of ticks and Coxiella burnetii will be done. The goals of the comparative genomics project are to characterize a core set of proteins for Coxiella spp. and to identify genes unique to C. burnetii that could facilitate this pathogen's transmission to or virulence in vertebrates.

Rationale

Q fever is a Category B select agent and has long been considered of high potential as an agent for biological warfare or terrorism. [2] Although infection mortality is low and secondary transmission between humans is rare, the acute illnesses frequently incapacitate infected individuals, and the attack rate can be high after point-source exposures. [3, 4] Moreover, in some cases acute illness is followed by chronic infection, such as "culture-negative" endocarditis, [3] or by a disabling post-infection fatigue syndrome. [5] Besides natural cycles of C. burnetii among wildlife and their tick vectors, the disease is enzootic and endemic in many regions of the world with cattle, goat, and sheep raising. The high concentrations of infectious microorganisms in placentas, milk, vaginal secretions, and feces of infected animals provide a ready, "low-tech" source of an infectious agent for would-be terrorists. Excreted organisms are highly resistant to drying, and the minimum inoculum for infection by the aerosol route is low. There have been several outbreaks of disease from point-source exposures through inadvertent aerosols, so sophisticated "weaponization" is not a requirement for successful delivery of this agent. Ampicillin-resistant C. burnetii have been obtained through genetic transformation with C. burnetii-E. coli shuttle plasmids, [5] so engineering for resistance to commonly recommended antibiotics, such as tetracyclines, is feasible.

Strain Selection

Diverse geographical distribution of C. burnetii has resulted in the accumulation of varied isolates that appear to be serologically similar. Isolates from mammalian and arthropod hosts have been differentiated based on genetic and phenotypic differences into at least 6 different genomic groups. [26-29] We propose to have a representative strain from each genomic group sequenced. The strains selected constitute a selection based on the virulence potential, disease phenotypes, host organism, geographical distribution, and gross genomic distinctions.

Biology of C. burnetti

A hallmark property of C. burnetii is its ability to remain infectious after extended exposure to extreme environmental circumstances (e.g. elevated temperature, desiccation, osmotic shock, ultraviolet light, and chemical disinfectants) [6]. This ability to survive a harsh intra- and extracellular environment may rely, in part, on a developmental life cycle with various forms specialized for propagation, surviving intracellular stress and persisting in the extracellular environment (in a non-replicating state) for long periods between hosts [9] [6].

In vitro, C. burnetii passively enters and replicates within a variety of epithelial, fibroblast, and macrophage-like cell lines [10]. Internalization into host cells occurs by a microfilamentdependent parasite-directed endocytic process [11]. The nascent parasite-containing phagosomes mature through the endocytic pathway, eventually acquiring the properties of secondary lysosomes. C. burnetii has an absolute requirement for a moderately acidic pH to activate metabolism and exploits the only intracellular niche capable of establishing such a pH [7, 8, 12]. The organism replicates to high numbers, albeit at a slow replication rate (~12 h doubling time) within this vacuole, despite the presence of toxic host factors, such as acid hydrolase, oxygen and nitrogen radicals, and defensins which are normally considered bactericidal [10].

C. burnetii is generally maintained in nature by cycles involving many species of animals, both herbivores and carnivores, as well as arthropods which become infected by taking a blood meal [13] [14]. Domestic animals are involved in the cycle of infection (cattle, sheep and goats are the primary reservoirs) and humans become infected via aerosols that originate primarily from contaminated animal products [15]. These infectious cycles provide ecological niches that might select for highly virulent genetic variants. Isolates obtained from a variety of wild vertebrates and arthropods have been classified into genomic groups by various methods and while these groups do not correlate with host, geographical location or date of isolation, the type of human disease caused by isolates in each group is similar, implying that specific isolate groups have a particular virulence potential in humans, determined by specific factors and molecular mechanisms encoded by each.

C. burnetii causes a wide spectrum of human disease. The disease manifestations of C. burnetii infection in humans can be separated into acute and chronic illnesses. Acute disease commonly presents as a flu-like illness with hallmark cyclic fever and periorbital headache [16]. Pneumonitis and hepatitis are frequent complications but acute disease is commonly self-limiting. Various antibiotics, including tetracyclines, are effective for abrogating acute disease [17]. Until recently, clinically, the illness fell within the group of FUO (fever of unknown origin) syndromes and was not commonly recognized or diagnosed. In many areas of the world, a high percentage of the population (10-20%) has serological evidence of previous infection [18]. In contrast, chronic infection is much less common and has a grim prognosis [19] [20]. Chronic disease most frequently manifests as endocarditis and hepatitis with recognition of these infections increasing worldwide. Recently a group of chronic infection patients, reported in Australia, presented with similar symptoms to chronic fatigue syndrome [21]. Chronic infections appear to be associated with a suppression of the cell-mediated immune system [22]. Chronic infections have not responded well to a variety of antibiotic regimens, with patients receiving a combination of doxycycline plus chloroquine, administered over 1-2 years, showing the best outcome [23]. Thus acute infection with C. burnetii leads to long-term vascular inflammation and points to latent infections comparable to that noted for Chlamydia pneumonia and other infectious agents. From a public health and bio-defense perspective, exposure to different strains, either natural, or due to illegitimate release, may have quite different outcomes. Exposure to acute disease isolates would likely result in predominantly flu-like illness with the potential for long term cardiovascular disease risk. Exposure of a population to chronic disease isolates would probably not result in significant immediate disease cases, but would likely result in chronic illnesses developing months or years from initial exposure.

Biology of R. grylli

Rickettsiella grylli is an obligate intracellular parasite of Gryllus bimaculatus, and related species of crickets. Members of the genus Rickettsiella are intracellular pathogens of invertebrates, including insects, crustaceans, and arachnids [24, 25]. The infectious forms are Gram-negative rod-shaped organisms that develop intracelluarly into larger vegetative forms, similar to the developmental cycle observed for Chlamydia spp and Coxiella. Bacterial replication takes place in cell vacuoles in the fat body, the hepatopancreas, and other organs of invertebrate hosts. Growth in cell-free media has not been demonstrated. They are pathogenic for their larval hosts and young and mature stages of invertebrate hosts, but of little virulence for vertebrates. The chief interest in Rickettsiellae stems from their infection of lab insectaries and other animal collections, as well as biocontrol for agricultural pests. They are maintained in the soil for years, and infection arises from contaminated soil rather than transovarian passage. The wide distribution of Rickettsiella geographically and in arthropod taxa suggests an early appearance in the course of evolution.

Since studies of host specificity, cultivation or antigenic analyses are not extensive, most of the knowledge is based on light and EM observations. The developmental cycle is comprised of a small, dense infectious particle, which is phagocytosed by the host cell, and within the intravacuolar compartment, it differentiates into less electron-dense intermediate- and large-sized forms that multiply. As the vacuole fills to capacity, large forms recondense to smaller dense particles that eventually escape the vacuole to start another infectious cycle. This developmental cycle is very similar to that described for C. burnetii involving 2-3 distinct morphological variants.

Virulence for invertebrate hosts other than host of isolation has been studied. R. grylli has been found to grow in Orthoptera, Lepidoptera and Coleoptera orders of insects, and Isopod and Amphipod crustaceans. R. grylli has also been shown to multiply in the mouse when large doses are inoculated by the intraperitoneal route or by inhalation, but the mouse overcomes infection and organisms are cleared in about 2 weeks.

Projects

Coxiella burnetii MSU Goat Q1

Coxiella burnetii Dugway 7E9-12

Coxiella burnetii RSA 331

Coxiella burnetii RSA 334

Rickettsiella grylli

Production Status

References

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